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oai:figshare.com:article/34017676 2026-09-28T17:52:50Z category_12 category_13 category_15 category_272 category_734 category_61 category_64 category_128 category_133 portal_5 item_type_1 month_year_09_2026

Cluster validations for larva glial annotations. Amanda A. G. Ferreira (25132828) Sergio Córdoba (645937) Raghuvanshi Rajesh (25132831) Ben Jiwon Choi (25132834) Claude Desplan (45436) Cell Biology Genetics Neuroscience Environmental Sciences not elsewhere classified Biological Sciences not elsewhere classified Developmental Biology Cancer Hematology Plant Biology innovative computational approach genetic markers expressed div >< p subcellular mrna compartmentalization optic lobe glia two large single detailed transcriptomic analysis identify mrna species glial diversity observed adult visual system glial cell bodies glial cell types understand glial development visual system cell mrna cell bodies glial types glial cells glia types adult stages cell class cell atlas neuropil glia chiasm glia work provides transcriptionally appear pupal stages helps explain gradually change drosophila </ distinct mrnas differentially localized developmental trajectories developmental single comprehensively annotated also present allowed us (A) Dot plot showing the expression levels of the genes used for the validation of each annotated cluster in L3. (B) Left, hierarchical tree showing cluster annotations after validations reveals 10 cell types (in bold text), excluding cells of central brain origin. Note that Chiasm glia was manually separated into Inner and Outer Chiasm subtypes. Right, unsupervised clustering of larval glial cells. (C–M) Immunostaining and FISH analysis of marker gene expression in the L3 optic lobe. Neuropil structure is revealed by N-cadherin (Ncad) or HRP staining (yellow), while Repo (magenta) is used to label glia nuclei in the left panels. Marker gene expression is shown using antibodies, lacZ reporters, protein tags or specific Gal4 lines driving UAS-nGFP expression (cyan). (C) rost-lacZ reporter (yellow) is only expressed in subperineurial glia. Anti-Svp antibody (cyan) labels both perineurial and subperineurial glia. (D) Optix (cyan) is present in satellite, epithelial, and marginal glia but not in subperineurial, outer chiasm, and medulla neuropil glia. (E) Dll (cyan) labels epithelial, marginal, lobula complex neuropil and medulla neuropil glia but not satellite, outer, and inner chiasm glia. (F) DAT-Gal4 (cyan) is expressed in the outer and inner chiasm glia. It is less frequently observed in the inner chiasm glia. (G) Hth (cyan) antibody is higher in inner chiasm than in outer chiasm glia. (H) Hand-Gal4 (cyan) is only expressed in medulla neuropil glia. (I) HCR RNA FISH probes against Cpr51A (yellow) and repo (magenta) mRNA. Hoechst antibody is shown in gray. Cpr51A is expressed in the chiasm and lobula complex glia. The image shows the standard deviation projection of three consecutive focal planes 2 µm apart. (J) GFP-tagged CG4288 (cyan) is expressed only in the cortex and lobula complex glia but not in the perineurial, medulla neuropil, and inner chiasm glia. Asterisk indicates Bolwig organ. (K) GFP-tagged alphaTub85E (cyan) is expressed in retina wrapping glia. (L) GFP-tagged Npc2b (cyan) is expressed only in cortex glia from the central brain. (M) Adgf-D (cyan) is expressed in the central brain glia. pg = perineurial glia, spg = subperineurial glia, sg = satellite glia, eg/mg = epithelial and marginal glia, mng = medulla neuropil glia, xgo = outer chiasm glia, xgi = inner chiasm glia, cxg = cortex glia, rwg = retina wrapping glia, cb-cxg = central brain cortex glia, lcng = lobula complex neuropil glia. Gal4-UAS (>>), GFP/LacZ from construct (::). Scale bar = 20 µm. The code used to generate the graphs can be found in <a href="https://zenodo.org/records/21890456" target="_blank">https://zenodo.org/records/21890456</a>. 2026-09-28T17:57:37Z Image Figure 10.1371/journal.pbio.3004015.g002 https://figshare.com/articles/figure/_p_Cluster_validations_for_larva_glial_annotations_p_/34017676 CC BY 4.0